BK polyomavirus (BKPyV) is ubiquitous in the human population, with >80% seropositivity, and establishes a lifelong, subclinical, persistent infection of the urinary tract. Even during periodic episodes of replication in healthy individuals, as evidenced by excretion of virus into the urine, there are no clinical symptoms. In transplant patients, however, viral replication is associated with significant morbidity and can sometimes be fatal. The incidence of polyomavirus-associated nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow transplant recipients has risen concomitant with rising numbers of transplants being performed and the development of more effective immunosuppressive drug regimens. Presently, there are no effective anti-viral treatments for BKPyV, and therefore the clinician is faced with the dilemma of reducing the dose of immunosuppressive drugs to allow the patient's immune system to battle the virus, which raises the risk of graft rejection, or relying on palliative care. There are two genetic forms of BKPyV, the archetype virus, which is the circulating form of the virus and can be isolated from urine of healthy persons and transplant recipients, and rearranged variants, which are almost exclusively isolated from transplant patients. Until recently, our understanding of BKPyV biology has been based on studies of rearranged variants, which can replicate in cell culture. Our laboratory's development of a system with which to study the archetype virus positions us to address important fundamental questions about persistence and reactivation, including how culturable replication variants arise. The aims of this project are (1) to characterize the molecular mechanisms that regulate archetype viral replication, which is dependent on relative levels of expression of viral early genes and a virally-encoded miRNA; (2) to examine the genetic mechanisms leading to the rise of rearranged variants, which differ from the archetype virus in the DNA sequence of their non-coding control region; and (3) to establish a cell culture model for viral persistence, with which we will characterize the viral and cellular factors governing this process. The long term goals of these studies are to dissect the mechanisms that regulate BKPyV replication and to identify steps in the viral life cycle that may be amenable to the development of new antiviral drugs. Because all known polyomaviruses studied to date also exhibit a similar life style of persistence and reactivation, these studies have broad implications for the entire virus family.